{"id":41087,"date":"2021-07-22T09:29:59","date_gmt":"2021-07-22T09:29:59","guid":{"rendered":"https:\/\/dnaera.com\/rs\/?p=41087"},"modified":"2023-04-17T09:21:58","modified_gmt":"2023-04-17T07:21:58","slug":"celogenomove-sekvenovanie-vs-genotypizacia","status":"publish","type":"post","link":"https:\/\/dnaera.com\/rs\/blog\/celogenomove-sekvenovanie-vs-genotypizacia\/","title":{"rendered":"Celogen\u00f3mov\u00e9 sekvenovanie vs. genotypiz\u00e1cia \u2013 z\u00e1le\u017e\u00ed na kvalite, nie na kvantite d\u00e1t"},"content":{"rendered":"\n

Deoxyribonukleov\u00e1 kyselina, v skratke DNA, je nosite\u013ekou genetickej inform\u00e1cie a nach\u00e1dza sa v jadre takmer ka\u017edej bunky v na\u0161om tele. M\u00f4\u017eeme si ju predstavi\u0165 ako \u201eknihu receptov\u201c, v ktorej jednotliv\u00e9 recepty predstavuj\u00fa g\u00e9ny. V\u010faka g\u00e9nom si vie bunka vyrobi\u0165 v\u00fdsledn\u00e9 produkty, \u010do s\u00fa vo v\u00e4\u010d\u0161ine pr\u00edpadov prote\u00edny. Tie v na\u0161om organizme n\u00e1sledne zabezpe\u010duj\u00fa r\u00f4zne \u00falohy, od stavebn\u00fdch, regula\u010dn\u00fdch, transportn\u00fdch a\u017e po mnoh\u00e9 \u010fal\u0161ie. Pokia\u013e sa v t\u00fdchto receptoch (g\u00e9noch) vyskytne nejak\u00e1 chyba, v\u00fdsledn\u00fd produkt m\u00f4\u017ee ma\u0165 in\u00e9 vlastnosti, pr\u00edpadne sa nemus\u00ed v\u00f4bec tvori\u0165. Tieto chyby ozna\u010dujeme ako \u201emut\u00e1cie\u201c a pr\u00e1ve tie s\u00fa vo ve\u013ekej miere zodpovedn\u00e9 za r\u00f4zne odli\u0161nosti v na\u0161om organizme.<\/p>\n\n\n\n

\u010co konkr\u00e9tne sa p\u00ed\u0161e v t\u00fdchto receptoch sa n\u00e1m podarilo odhali\u0165 a\u017e za\u010diatkom tohto storo\u010dia, kedy vedci pre\u010d\u00edtali cel\u00fd \u013eudsk\u00fd gen\u00f3m. Ten sa sklad\u00e1 z pribli\u017ene 3 mili\u00e1rd stavebn\u00fdch blokov, ktor\u00e9 ozna\u010dujeme ako tzv. \u201enukleotidy\u201c. Cel\u00e1 sekvencia DNA je poskladan\u00e1 zo 4 z\u00e1kladn\u00fdch nukleotidov \u2013 aden\u00edn, guan\u00edn, cytoz\u00edn a tym\u00edn. Z tohto d\u00f4vodu sa pri sekvencii DNA stretneme s p\u00edsmenami A, G, C a T. Samotn\u00fd sled p\u00edsmen n\u00e1m v\u0161ak ni\u010d nepovie. Dnes u\u017e vieme, \u017ee g\u00e9ny tvoria len pribli\u017ene 2 % \u013eudsk\u00e9ho gen\u00f3mu a m\u00e1me ich pribli\u017ene 20 000. Zvy\u0161ok viac-menej nepozn\u00e1me, aj ke\u010f vieme, \u017ee aj v tejto \u010dasti sa nach\u00e1dzaj\u00fa d\u00f4le\u017eit\u00e9 oblasti pre spr\u00e1vne fungovanie na\u0161ich buniek.<\/p>\n\n\n\n

Ako ve\u013emi sa z poh\u013eadu genetiky navz\u00e1jom l\u00ed\u0161ime? Na\u0161a DNA sa medzi nami zhoduje a\u017e do miery 99,9 %. Na prv\u00fd poh\u013ead sa m\u00f4\u017ee zda\u0165, \u017ee sa takmer nel\u00ed\u0161ime. Ke\u010f sa v\u0161ak vr\u00e1time k ve\u013ekosti n\u00e1\u0161ho gen\u00f3mu, zist\u00edme, \u017ee sa l\u00ed\u0161ime v pribli\u017ene 3 mili\u00f3noch nukleotidov. A pr\u00e1ve tieto miesta, na ktor\u00fdch sa v popul\u00e1cii nach\u00e1dzaj\u00fa r\u00f4zne p\u00edsmen\u00e1, s\u00fa obrovsk\u00fdm z\u00e1ujmom mnoh\u00fdch \u0161t\u00fadi\u00ed. V\u010faka t\u00fdmto \u0161t\u00fadi\u00e1m dnes vieme napr\u00edklad poveda\u0165, \u017ee pokia\u013e sa u v\u00e1s vyskytuje na konkr\u00e9tnom mieste v DNA nejak\u00e9 konkr\u00e9tne p\u00edsmeno, potom m\u00e1te zv\u00fd\u0161en\u00fa genetick\u00fa predispoz\u00edciu na rozvoj nejak\u00e9ho ochorenia, ako je napr\u00edklad melan\u00f3m alebo infarkt myokardu.
V\u010faka obrovsk\u00e9mu pokroku v oblasti \u201e\u010d\u00edtania\u201c DNA m\u00e1 dnes takmer ka\u017ed\u00fd mo\u017enos\u0165 nahliadnu\u0165 priamo do svojich buniek, do sekvencie svojej DNA. Je to naozaj fenomen\u00e1lny \u00faspech vedy, ke\u010f\u017ee osekvenova\u0165 jeden \u013eudsk\u00fd gen\u00f3m st\u00e1lo e\u0161te na za\u010diatku tohto storo\u010dia pribli\u017ene 3 miliardy dol\u00e1rov, a trvalo to 13 rokov. Dnes sme sa u\u017e dostali pod 1000 dol\u00e1rov za gen\u00f3m a samotn\u00e9 sekvenovanie trv\u00e1 len nieko\u013eko min\u00fat.<\/p>\n\n\n\n

\"Cena<\/figure>\n\n\n\n

V s\u00fa\u010dasnosti s\u00fa vyu\u017e\u00edvan\u00e9 hlavne dva sp\u00f4soby \u010d\u00edtania DNA \u2013 sekvenovanie a genotypiz\u00e1cia.<\/p>\n\n\n\n

Sekvenovanie<\/h2>\n\n\n\n

Jednou z techn\u00edk vyu\u017e\u00edvan\u00fdch na stanovenie sekvencie DNA je tzv. sekvenovanie, teda ur\u010dovanie poradia nukleotidov v gen\u00f3me jednotlivca. V s\u00fa\u010dasnosti sa vyu\u017e\u00edvaj\u00fa najm\u00e4 dva pr\u00edstupy, a to tzv. celoex\u00f3mov\u00e9 a celogen\u00f3mov\u00e9 sekvenovanie.<\/p>\n\n\n\n

Celoex\u00f3mov\u00e9 sekvenovanie<\/strong><\/h4>\n\n\n\n

Technol\u00f3gia celoex\u00f3mov\u00e9ho sekvenovania je zameran\u00e1 len na konkr\u00e9tne miesta v gen\u00f3me, ktor\u00e9 poskytuj\u00fa inform\u00e1cie na v\u00fdrobu prote\u00ednov. Predpoklad\u00e1 sa, \u017ee tieto \u010dasti, naz\u00fdvan\u00e9 ako \u201eex\u00f3ny\u201c, tvoria pribli\u017ene 1 % gen\u00f3mu \u010dloveka. Spolo\u010dne v\u0161etky ex\u00f3ny v gen\u00f3me s\u00fa zn\u00e1me ako \u201eex\u00f3m\u201c, a preto je ich sp\u00f4sob sekvenovania zn\u00e1my ako celoex\u00f3mov\u00e9 sekvenovanie. Tento pr\u00edstup umo\u017e\u0148uje identifikova\u0165 mut\u00e1cie v k\u00f3duj\u00facej oblasti ak\u00e9hoko\u013evek g\u00e9nu, a nie iba v nieko\u013ek\u00fdch vybran\u00fdch g\u00e9noch. A ke\u010f\u017ee sa v\u00e4\u010d\u0161ina zn\u00e1mych mut\u00e1ci\u00ed sp\u00f4sobuj\u00facich ochorenie vyskytuje pr\u00e1ve v ex\u00f3noch, tento sp\u00f4sob sekvenovania je efekt\u00edvny v identifik\u00e1cii tak\u00fdchto potenci\u00e1lnych mut\u00e1ci\u00ed.<\/p>\n\n\n\n

Celogen\u00f3mov\u00e9 sekvenovanie<\/strong><\/h4>\n\n\n\n

Je v\u0161ak zn\u00e1me, \u017ee aj mut\u00e1cie mimo oblast\u00ed ex\u00f3nov m\u00f4\u017eu ovplyvni\u0165 g\u00e9nov\u00fa aktivitu a produkciu bielkov\u00edn, a t\u00fdm p\u00e1dom m\u00f4\u017eu vies\u0165 k r\u00f4znym ochoreniam. Pr\u00e1ve preto je najide\u00e1lnej\u0161\u00edm sp\u00f4sobom celogen\u00f3mov\u00e9 sekvenovanie, ktor\u00e9 ur\u010duje poradie takmer v\u0161etk\u00fdch nukleotidov v gen\u00f3me jednotlivca.<\/p>\n\n\n\n

V s\u00fa\u010dasnosti je najbe\u017enej\u0161ou pou\u017e\u00edvanou met\u00f3dou tzv. ILLUMINA sekvenovanie, v\u010faka ktor\u00e9mu vzniklo viac ako 90 % v\u0161etk\u00fdch sekvena\u010dn\u00fdch d\u00e1t na svete. N\u00e1zov tejto met\u00f3dy odkazuje na americk\u00fa spolo\u010dnos\u0165, ktor\u00e1 sa podie\u013ea na v\u00fdvoji, v\u00fdrobe a predaji syst\u00e9mov na anal\u00fdzu genetick\u00fdch vari\u00e1ci\u00ed a biologick\u00fdch funkci\u00ed, a ktor\u00e1 t\u00fato met\u00f3du komercionalizovala. T\u00e1to met\u00f3da je zalo\u017een\u00e1 na detegovan\u00ed jednotliv\u00fdch nukleotidov (A, G, C a T) potom, ako sa za\u010dlenia do rast\u00faceho vl\u00e1kna DNA. Detekcia nukleotidov je zalo\u017een\u00e1 na fluorescen\u010dnom sign\u00e1li, to znamen\u00e1, \u017ee ka\u017ed\u00fd nukleotid je ozna\u010den\u00fd nejakou fluorescen\u010dnou zna\u010dkou (m\u00f4\u017eeme si ju predstavi\u0165 ako nejak\u00fa farebn\u00fa zna\u010dku) a po pridan\u00ed sa do re\u0165azca po\u010d\u00edta\u010d vyhodnot\u00ed dan\u00fd sign\u00e1l.<\/p>\n\n\n\n

Sta\u010d\u00ed v\u0161ak pre\u010d\u00edta\u0165 cel\u00fd gen\u00f3m len raz?<\/h2>\n\n\n\n

Moment\u00e1lne e\u0161te nie sme v bode, kedy by sta\u010dilo pre\u010d\u00edta\u0165 cel\u00fd \u013eudsk\u00fd gen\u00f3m len jedenkr\u00e1t. V s\u00fa\u010dasnosti toti\u017eto e\u0161te nie s\u00fa tak presn\u00e9 sekvena\u010dn\u00e9 met\u00f3dy, aby dok\u00e1zali len po\u010das jedn\u00e9ho \u010d\u00edtania pre\u010d\u00edta\u0165 bezchybne cel\u00fd gen\u00f3m. Pr\u00e1ve z tohto d\u00f4vodu odpor\u00fa\u010da spolo\u010dnos\u0165 ILLUMINA minim\u00e1lne pokrytie 30x. To znamen\u00e1, \u017ee v priemere je ka\u017ed\u00fd nukleotid pre\u010d\u00edtan\u00fd nez\u00e1visle 30-kr\u00e1t.<\/p>\n\n\n\n

Niektor\u00e9 spolo\u010dnosti v\u0161ak dnes pon\u00fakaj\u00fa aj celogen\u00f3mov\u00e9 sekvenovanie s ove\u013ea men\u0161\u00edm pokryt\u00edm, v niektor\u00fdch pr\u00edpadoch dokonca s pokryt\u00edm len 1x. Pri sekvenovan\u00ed s tak\u00fdm n\u00edzkym pokryt\u00edm je v s\u00fa\u010dasnosti nere\u00e1lne pre\u010d\u00edta\u0165 cel\u00fd gen\u00f3m. Ako je potom mo\u017en\u00e9 posklada\u0165 cel\u00fa sklada\u010dku, ke\u010f z \u010d\u00edtania nie s\u00fa k dispoz\u00edcii niektor\u00e9 dieliky? <\/p>\n\n\n\n

Tento proces je pomerne zlo\u017eit\u00fd, ale v jednoduchosti sa vyu\u017e\u00edva princ\u00edp tzv. \u201eimput\u00e1cie\u201c. T\u00fdmto sp\u00f4sobom sa \u0161tatisticky doplnia nepre\u010d\u00edtan\u00e9 miesta, a to na z\u00e1klade zn\u00e1mych haplotypov v popul\u00e1cii. Vyu\u017e\u00edva sa tu to, \u017ee jednotliv\u00e9 nukleotidy sa nededia samostatne, ale dedia sa spolo\u010dne v skupin\u00e1ch, ktor\u00e9 ozna\u010dujeme ako \u201ehaplotypy\u201c. Aby sme v\u0161ak mohli pova\u017eovat sekvenovanie s tak\u00fdmto n\u00edzkym pokryt\u00edm za relevantn\u00e9, je potrebn\u00e9 ma\u0165 ve\u013ek\u00fd a popula\u010dne \u0161pecifick\u00fd referen\u010dn\u00fd panel, na z\u00e1klade ktor\u00e9ho bude mo\u017en\u00e9 doplni\u0165 ch\u00fdbaj\u00face dieliky gen\u00f3mu jednotlivca s vysokou presnos\u0165ou. Av\u0161ak aj pri splnen\u00ed t\u00fdchto podmienok nie je tento pr\u00edstup vhodn\u00fd na anal\u00fdzu ve\u013emi vz\u00e1cnych genetick\u00fdch variantov.
<\/p>\n\n\n\n

Sekvenovanie \u0161pecifick\u00fdch g\u00e9nov<\/h2>\n\n\n\n

V klinickej praxi sa \u010dastokr\u00e1t nesekvenuje cel\u00fd gen\u00f3m, ani ex\u00f3m, ale sekvenuj\u00fa sa len konkr\u00e9tne vybrat\u00e9 \u00faseky \u2013 konkr\u00e9tne g\u00e9ny. Toto diagnostick\u00e9 vy\u0161etrenie je \u00fa\u010dinn\u00fdm n\u00e1strojom potvrdenia alebo vyvr\u00e1tenia nejakej \u0161pecifickej patog\u00e9nnej mut\u00e1cie ved\u00facej k nejak\u00e9mu z\u00e1va\u017en\u00e9mu ochoreniu, ktor\u00e9 sa vo v\u00e4\u010d\u0161ej miere vyskytuje v rodinnej hist\u00f3rii dan\u00e9ho pacienta. <\/p>\n\n\n\n

Genotypiz\u00e1cia<\/h2>\n\n\n\n

Napriek tomu, \u017ee n\u00e1m celogen\u00f3mov\u00e9 sekvenovanie poskytuje najlep\u0161\u00ed obraz o na\u0161om gen\u00f3me, st\u00e1le patr\u00ed medzi relat\u00edvne drah\u00e9 met\u00f3dy. Pr\u00e1ve preto sa v s\u00fa\u010dasnosti v komer\u010dn\u00fdch DNA testoch vyu\u017e\u00edva najm\u00e4 pr\u00edstup tzv. genotypiz\u00e1cie, a to z d\u00f4vodu jej efektivity v pomere cena\/v\u00fdkon. Ide o anal\u00fdzu konkr\u00e9tnych miest v DNA na \u010dipe. Na rozdiel od celogen\u00f3mov\u00e9ho sekvenovania sa teda ne\u010d\u00edta cel\u00e1 DNA, ale len konkr\u00e9tne miesta, ktor\u00e9 boli vybrat\u00e9 na z\u00e1klade toho, \u010di o nich nie\u010do pozn\u00e1me a teda \u010di ich vieme s nie\u010d\u00edm asociova\u0165. Tieto miesta zah\u0155\u0148aj\u00fa napr\u00edklad mut\u00e1cie sp\u00e1jan\u00e9 s r\u00f4znymi ochoreniami alebo s atletick\u00fdm potenci\u00e1lom. Viac o tom, ako prebieha genotypiz\u00e1cia, si m\u00f4\u017eete pre\u010d\u00edta\u0165 v tomto blogu<\/a>.<\/p>\n\n\n\n

Ktor\u00fd pr\u00edstup je teda v s\u00fa\u010dasnosti vhodnej\u0161\u00ed pre komer\u010dn\u00e9 genetick\u00e9 testovanie?<\/h2>\n\n\n\n

Ako bolo u\u017e spomenut\u00e9 vy\u0161\u0161ie v texte, samotn\u00fd sled nukleotidov v gen\u00f3me n\u00e1m ni\u010d nepovie, k\u013e\u00fa\u010dov\u00e1 je asoci\u00e1cia konkr\u00e9tnych zmien s konkr\u00e9tnymi znakmi.

Celogen\u00f3mov\u00e9 sekvenovanie s\u00edce poskytuje najucelenej\u0161\u00ed poh\u013ead na gen\u00f3m jednotlivca, av\u0161ak mus\u00ed by\u0165 uroben\u00e9 kvalitne, s dostato\u010dne ve\u013ek\u00fdm pokryt\u00edm. To je v\u0161ak st\u00e1le pomerne n\u00e1kladn\u00e9, a preto v kombin\u00e1cii s t\u00fdm, \u017ee v s\u00fa\u010dasnosti nepozn\u00e1me funkciu v\u00e4\u010d\u0161iny miest v gen\u00f3me, nem\u00f4\u017eeme dnes pova\u017eova\u0165 tento pr\u00edstup za efekt\u00edvny pre komer\u010dn\u00e9 genetick\u00e9 testovanie.

Pri genotypiz\u00e1cii sa neanalyzuje cel\u00fd gen\u00f3m, sleduj\u00fa sa len vybrat\u00e9 miesta so zn\u00e1mou funkciou. Tento efekt\u00edvny pr\u00edstup umo\u017enil zn\u00ed\u017ei\u0165 cenu za genetick\u00e9 testovanie nato\u013eko, \u017ee sa stal vhodn\u00fdm n\u00e1strojom pre komer\u010dn\u00e9 vyu\u017eitie. Pr\u00e1ve preto vyu\u017e\u00edvame tento pr\u00edstup v na\u0161ich DNA testoch aj my v DNA ERA.

Zdroje<\/strong><\/p>\n\n\n\n

  1. Li, Y., Willer, C., Sanna, S., & Abecasis, G. (2009). Genotype imputation. Annual review of genomics and human genetics, 10, 387-406.<\/li>
  2. Shendure, J., Balasubramanian, S., Church, G. M., Gilbert, W., Rogers, J., Schloss, J. A., & Waterston, R. H. (2017). DNA sequencing at 40: past, present and future. Nature, 550(7676), 345-353.<\/li>
  3. https:\/\/www.illumina.com\/science\/technology\/next-generation-sequencing\/plan-experiments\/coverage.html<\/li><\/ol>\n\n\n\n

    <\/p>\n\n\n\n

    Zdroje fotografi\u00ed<\/strong><\/p>\n\n\n\n

    1. https:\/\/www.freepik.com\/photos\/technology’>Technology photo created by freepik – www.freepik.com<\/a><\/li>
    2. https:\/\/en.wikipedia.org\/wiki\/$1,000_genome#\/media\/File:Cost_per_Genome.png<\/a><\/li><\/ol>\n","protected":false},"excerpt":{"rendered":"

      Deoxyribonukleov\u00e1 kyselina, v skratke DNA, je nosite\u013ekou genetickej inform\u00e1cie a nach\u00e1dza sa v jadre takmer ka\u017edej bunky v na\u0161om tele. M\u00f4\u017eeme si ju predstavi\u0165 ako \u201eknihu receptov\u201c, v ktorej jednotliv\u00e9 recepty predstavuj\u00fa g\u00e9ny. V\u010faka g\u00e9nom si vie bunka vyrobi\u0165 v\u00fdsledn\u00e9 produkty, \u010do s\u00fa vo v\u00e4\u010d\u0161ine pr\u00edpadov prote\u00edny. Tie v na\u0161om organizme n\u00e1sledne zabezpe\u010duj\u00fa r\u00f4zne \u00falohy, […]<\/p>\n","protected":false},"author":6,"featured_media":41089,"comment_status":"closed","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"_acf_changed":false,"content-type":"","inline_featured_image":false,"footnotes":""},"categories":[1],"tags":[58,59],"place-taxonomy":[],"class_list":["post-41087","post","type-post","status-publish","format-standard","has-post-thumbnail","hentry","category-uncategorized-sk","tag-dna-analyza","tag-dna-test"],"acf":[],"views":3338,"_links":{"self":[{"href":"https:\/\/dnaera.com\/rs\/wp-json\/wp\/v2\/posts\/41087","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/dnaera.com\/rs\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/dnaera.com\/rs\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/dnaera.com\/rs\/wp-json\/wp\/v2\/users\/6"}],"replies":[{"embeddable":true,"href":"https:\/\/dnaera.com\/rs\/wp-json\/wp\/v2\/comments?post=41087"}],"version-history":[{"count":3,"href":"https:\/\/dnaera.com\/rs\/wp-json\/wp\/v2\/posts\/41087\/revisions"}],"predecessor-version":[{"id":1352443,"href":"https:\/\/dnaera.com\/rs\/wp-json\/wp\/v2\/posts\/41087\/revisions\/1352443"}],"wp:featuredmedia":[{"embeddable":true,"href":"https:\/\/dnaera.com\/rs\/wp-json\/wp\/v2\/media\/41089"}],"wp:attachment":[{"href":"https:\/\/dnaera.com\/rs\/wp-json\/wp\/v2\/media?parent=41087"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/dnaera.com\/rs\/wp-json\/wp\/v2\/categories?post=41087"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/dnaera.com\/rs\/wp-json\/wp\/v2\/tags?post=41087"},{"taxonomy":"place-taxonomy","embeddable":true,"href":"https:\/\/dnaera.com\/rs\/wp-json\/wp\/v2\/place-taxonomy?post=41087"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}